**3.1.1.2 Fermentation medium and experimental system**

A solution of dried permeate from UF whey permeate from the Dairy Plant in Wolsztyn, Poland, was used as a substrate in this study. The solution was prepared by dissolving dried permeate in distilled warm water to obtain 50 g L-1 lactose concentration in wastewater, while the initial COD was 56 g L-1.

Fermentation process was carried out in three UASB reactors with an active volume of 5 L. There were the gas-liquid-solid (G-L-S) separators on the top of each reactor. Whey permeate solution was pumped continuously to the bottom part of the reaction tank by means of the peristaltic pump. The necessary mixing was achieved through the upward wastewater flow and a stirrer operated at 40 rpm. The reactors were water-jacketed and operated at a constant temperature of 25ºC ± 1°C. The pH of mixed liquid in the reactors was controlled automatically at pH 4.76 – 4.86 with 2 M NaOH.

For start-up of continuous culture, 1 L of the beads culture medium were grown at 25C for 24 h in a 2 L Erlenmeyer flask filled with 0.5 L of UF whey permeate after heat sterilization (120C, 20 min). The concentration of lactose in whey permeate was 50 g L-1. Mixing was achieved by stirring with a magnetic stirrer at 200 rpm. The cell suspension was then aseptically transferred to each UASB reactor which was kept in batch operation for 24 h before switching on the continuous feeding. The reactors were operated at the HRTs of 12, 24 and 48 h. At each HRT the reactors were operated till they had reached the steady-state (the steady-state conditions were evidenced when the standard deviations of the ethanol concentrations and lactose concentrations in the effluent distillate were within 3%).
